ATRIBUCIONES Y DEBERES DEL PRESIDENTE DE LA REPÚBLICA

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Figure 1 5 Growth and enterotoxin B produ ction by strai n S-6 in a fermenter in CH medium at uncontro lled pH (open symbols ) and p H 6.5 (closed symbols) . OD, c.; enterotoxin B , o; pH, o.

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s l ight l y h i gher at c ontr o l l e d pH as � ompar ed with produc t i on vJhen pH Has not c ontrol l e d in the f erment er , w i th an optimum ·

pH of 8 . 0 .

Th e spec i fic rat e of ent er o t oxin C produc t i on under any

c ondi t i ons of pH in the f erm enter was extremely low ( b etwe en 0 . 05 and 0 . 1 3 pg/unit c e l l mass/h ) as c ompar ed with $hake­ flasks ( 0 . 83 ) , with an opt imum pH o f 8 . 0 in the ferment er . Ther e wa s very l i t t l e differ enc e in �r owth patt ern b etween

that wh en the pH was not c ontroll ed , and at pH 6 . 5 , 7 . 3 and

8 . 0 .

Eff e c t o f on other extr ac e l lular The produc t i on of l ipas e , de oxyrib onuc l eas e , lys o zyme and TEP during 1 2 h incub a t i on in the ferment er was higher at c ontr o l l ed pH than at unc ont r o l l e d pH , exc ept for deoxyrib onuc l eas e produc t i on by s tr a in S-6 ( Tab l e 1 0 ) . Y.iaximv.m yi e lds were generally ob t ained b etwe en 6 . 5 and 7 . 3 , b ut the opt imum p H vari ed c on­

s iderab l y b e tv1 e en strains .

3 . 3 . 7 �ffec t of pH on th e of ent erot ox ins and o ther extrac e l lular in LA m e d itun

I t had b e en found in pr e l iminary shake-flask exp eriments ( S ec t i on 2 . 3 . 3) that in .u.A m edium , the produc t i on of ent ero­ toxin A b y s train 1 00 was s imilar to that in CH medium . bnt erot oxin B produc t i on in AA medium was l es s than in CH medium , b ut s t i l l app ear ed to b e adequat e for further exp eri­ ments . The produc t i on o f ent erotoxin C , which in th e

f erment er in CH medium had b e en extremely l ow , was hi ghe r

i n AA m edium than i n CH m e dium i n shake-flasks . Growing

the s t aphyloc occal s trains in AA medium in the fermenter a l s o had the great advantage over CH medium that v ery l i t t l e f o aming oc curre d and c ons equently antifoam was not ne eded. , apart from an ini t ial 0 . 3 mg/ml as d e s crib ed in Sec ti on 3 . 2 . 1 . The

pr oduc t i on o f ent erotoxin in AA m edium was there for e d e t ermined at varying pH values , with the partial pr e s s ur e of d i s s olved oxygen at b e tw e en 30 and 40 mm Hg .

Effe c t o f on ent erot oxin A When the pH was

not h e l d c ons t ant , strain 1 00 grew mor e s l owly in AA than in CH m edium with a c orr e s p onding l es s er inc reas e in pH , and

66 .

and the final yi eld of ent erotoxin

A

\.vas c ons iderab l y h i c.;her

in th e ( Fi g . 1 6 ) . ·nowever , the spec i f ic rat e o f

ent erot oxin produc tion in the f ermenter v1i thout pH c ontr o l was 0 . 1 8 pg/uni t c e l l mass/h i n AA medium , a s c omp ar e d with 0 . 3 9 in CH medium , in d i c a t inG that the higher f ina l yi e l d of

ent erot oxin A in AA m edium was due t o the change in grO\·Jth

patt ern result ing in c ontinued ent erotoxin produc t i on . The produc t i on o f cri t erotoxin A by strain 1 00 was hi gh e s t under c ontro l l ed pH c ondi t i ons i n AA medium, with an optim1illl pH o f 6 . 5 in agr e ement with the findings in CH medium . The p'att ern of gr o'.·:th was s imilar in the f erm ent er under a l l pH c ondi ti ons . The spec ific rat e of ent er ot oxin produc t i on

( qp in Tab l e 1 1 ) indicates that the variat ion in yi e l ds o f ent erot oxin A under different pH c ondi t i ons in AA m edium v.'as due to a di fferenc e in the s p ec ific rat e of ent erot oxin pro­ duc t i on .

Eff e c t of pH on ent ero toxin J3 "Pr oduc t i on . rrh e produc t i on of ent erotoxin B by s train

S-6

during 1 2 h in the ferm ent er �as l ovJ er in AA medililll und er all c o.rid i t i ons o f pH . Ho;,-..rever , the s pec ific rat e s of ent erot oxin produc t i on v1er e higher in iu"l medium _ ( qp in Table 1 1 ) , than in CH rnedi1rrn ( qp in T ab l e 1 0 ) o

In AA medium , the yi e ld and s p e c i fic rat es o f ent erotoxin produc tion w er e both high e s t at c ontrolled pH as c ompared wi th unc ontr o l l ed pH . 'I'he hi ghes t yi eJ_d during 1 2 h was ob tained at a pH of b e t w e en 6 . 5 and 7 - 3 whi l e the opt imum pH for the spec i fi c rat e of ent erot oxin produc t i on was

6 . 5 .

Effec t of on ent erot oxin C The yi e l d and

spec i fic rat e s of ent ero toxin C produc t i on by strain 361 were much higher in AA m edium in the shake-flask and f erment er , under all c onditions of pH . In AA medium , the ent erot oxin yi e l d was highest under c ontr o l l e d c ond itions o f pH , with an optimum pH o f 7 . 3 . The spec i fi c rat es of ent er o t oxin C

pr oduc tion ( q in Tab l e _p 1 1 ) were a l s o higher at c ontr o l l e d pH _'

with an opt imum pH o f 6 . 5 .

Eff e c t o f on other extrac e l lul ar The produc - t i on of l i pas e , deoxyrib onuc l e as e , lys ozym e and TEP was generally higher under c ondi t i ons of c ontr o l l ed pH than at

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Hours of I ncubation

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Figure 1 6 Growth and enterotoxin A production by strain 1 00 in a fermenter in CH medium (open symbols ) and AA medium (closed symbols ) .

OD, 6; enterotoxin A , o ; pH, o . 67.

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TABLE 1 1 .

S train and

ent erot oxin type 1 00 A 1 00 A 1 00 A 1 00 A 1 00 A S-6 B S-6 B S-6 B S-6 B S-6 B S-6 B 36'1 c 361 c 361 c 361 c 361 c

Effec t of pH on the produc ti ons o f ent ero toxins A , B and C and other extrac e l lular proteins in AA medi� .

l.J e oxyrib o-

Lipas e Lys o z yme TEP :Final

pH _{(pg/ml ) ( qp ) ( uni t s /ml )} nuc l eas e _{( uni ts/ml ) ( mg/ml ) OD}

( units/ml ) SF a 7

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27 8 0 . 1 0 . 1 4 1 8 NCb 20 0 . 1 8 20 1 3 0 0 . 1 6 24 6 . 0 2L� 0 . 20 5 1 7 0 0 . 06 27 6 . 5 3 2 0 . 25 5 10 0 . 2 ·0 .05 27 7 - 3 20 0 . 1 8 48 1 5 0 0 . 1 5 25 BFa 1 94 1 . 54 59 86 . 0 . 1 0 . 68 1 7 NCb , c 97 1 . 70 1 00 84 0 . 2 0 . 45 26 6 . 0c 96 1 . 83

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38 . 28 0 . 2 0 . 46 3 5 6 . 5c 1 29 2 . 1 2 240 37 0 . 3 0 . 67 25 7 . 3 c 1 31 1 . 91 '1 1 0 47 0 '1 . 1 2 23 8 . 0 78 '1 . 02 '1 1 4 1 25 0 . 1 0 . 46 17 SF a '1 00 1 . 20 67 1 0 1 . '1 0 . 64 24 NCb 3 5 0 . 5 5 9 0 1 9 1 . 1 0 . 53 28 6 . 0 25 0 . 34 48 1 2 0 . 9 0.21 1 7 6 . 5 53 0 . 71 320 8 3 . 1 1 . 02 23 7 - 3 30 0 . 3 7 3 20 1 9 3 . 0 0 . 90 26

a SF indicates shake-flask cultures . Al l other exp erime�t s carri ed out in the f erment er . b NC indicat es pH was not c ontro l l e d .

c Strain S - 6 al s o produc ed ent er o t oxin A at unc ontrol l ed pH ( 2 pg/ml ) , pH 6 . 0 ( 3 pg/ml ) ;

pH 6 . 5 ( 2 pg/ml ) and pH 7 . 3 ( 2 f-g/ml ).

m (X)

69 . unc ontro l l ed pH ( Tab l e 1 1 ) . Alth ouBh there was a c ons i der-

ab l e s train var i c::.t i on , m G.ximum yi e lds of th es e extrac e l lular pro t eins w e r e ob tained b e t w e en pH 6 .

5

and 7 . 3 in mo s t c a s es . 3 . 3 . 8 Eff e c t o f gas f l ow on c e l l s i n AA

medium

I n ini t i a l exp erim ent s vvi th A A ri1 eclium in the f erment er , the s am e aera t i on pr o c e dur e was a d opt ed as for CH m edium , i . e . air and

N2 ( 2

l i t r e s per min ) w e r e turned on and t h e oxygen t i trat or a d j us t ed s o tha t the part i a l pres s ur e of d i s s o lved

oxye;en was maintained at 30 -40 mm H g . Ilov1ever , as l i t t l e f o aming oc c urr ed in AA medium, n o ant i f oam vms adde d , wher eas 0 . 3 mg/ml ant i f o am was add ed ini t ia l l y to CH medium . I t was

f ound vv-i th s train S-6 that th er e 'dO. S a lag p er i o d o f approxi­ mat e ly 1 0 h . Kub i t s c h ek ( 1 9 / 0 ) r eported that a c u l t ur e may

f a i l to grow in a c hemo s t a t if i t i s aerat ed immedi a t e l y . 'rher e f or e , a e r a t i on was d el ayed m1t i l gro1·1th \·..ras e s t ab l i s h ed

and OD r e ached approxim a t ely 1 . 0 , and thi s gr e a t l y r e duc ed the l ag peri o d f o r s tra in S-6 .

H owever , when strain 1 00 vm s gr o1·m 1n AA m edium in the ·

f erm ent er , there was inme diat e lys is o f c e l l s , anG. the OD

r apidly d e c r e a s e d ( Fi g . 1 7 ) . Thi s e ffect was extr emely r epro ­ duc ib l e , and was n o t p e culiar t o s train 1 00 e I f s train S - 6 w a s subj e c t t o a n air and N 2 f l m-.r o f 4 l i t r e s p er min as s o on as the f erment er had b e en inocul a t e d , a cul tur e w i t h an ini t i al

OD o f 0 . 20 was r educ ed to 0 . 01 in 4 h. Shak e - f l ask exo eri­ m ents showed that c el l s taken from t h e ferment er were c aoab l e

o f normal grmvth , and that th e medium did not c on t ain an inhib i t or . I t has b e en r eport e d ( Kubitschek , 1 970 ) that in order t o a dapt t o chemos t a t c ul tur e s ome c e l l s r e q uir e

co2 •

The ni t r o g en in the air and N 2 mixtur e was ther e f o r e r eplac e d w i t h

co2 ,

b u t t h i s d i d n o t pr event lys is o f th e s taphyloc oc c al c el l s . Lys i s was not d e c r ea s e d by using an ino c u l um of

younger c e l l s or of higher c e ll d ensi ty.

Exp eriment s with s trains 1 00 and S-6 in whi c h air ( 0 . 3 l i t r e s p er min ) w a s bubb l e d through c u l tur es in b o i l ing t ub es showed that it was pos s ib l e t o lys e c e l l s vrhich had b e en grown in

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